Journal of Invertebrate Pathology 104 (2010) 44–50

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Journal of Invertebrate Pathology

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Dose dependency of time to death in single and mixed infections with a wildtypeand egt deletion strain of Helicoverpa armigera nucleopolyhedrovirus

Liljana Georgievska a,c, Kelli Hoover a,b,*, Wopke van der Werf c, Delia Muñoz d, Primitivo Caballero d,Jenny S. Cory a,e, Just M. Vlak a

a Wageningen University, Laboratory of Virology, Binnenhaven 11, Wageningen 6709 PD, The Netherlandsb Pennsylvania State University, Department of Entomology, 501 Ag Sciences & Industries Building, University Park, PA 16802, USAc Wageningen University, Centre for Crop Systems Analysis, P.O. Box 430, 6700 AK Wageningen, The Netherlandsd Entomología Agrícola y Patología de Insectos, Departamento de Producción Agraria, Universidad Pública de Navarra, Campus Arrosadía, 31006 Pamplona, Spaine Simon Fraser University, Department of Biological Sciences, 8888 University Drive, Burnaby, BC, Canada V5A 1S6

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 July 2009Accepted 27 January 2010Available online 1 February 2010

Keywords:Helicoverpa armigeraHaSNPVGenetically modified baculovirusMixed infectionDose range

0022-2011/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.jip.2010.01.008

* Corresponding author. Address: Pennsylvania StaEntomology, 501 Ag Sciences & Industries Building, UnFax: +1 814 865 3048.

E-mail address: kxh25@psu.edu (K. Hoover).

Recombinant insect nucleopolyhedroviruses lacking the egt gene generally kill their hosts faster thanwild-type strains, but the response of insects to mixtures of virus genotypes is less well known. Here,we compared the survival time, lethal dose and occlusion body yield in third instar larvae of Helicoverpaarmigera (Hübner) after challenge with wild-type H. armigera SNPV (HaSNPV-wt), a strain with a deletionof the egt gene, HaSNPV-LM2, and a 1:1 mixture of these two virus strains. A range of doses was used todetermine whether the total number of OBs influenced the response to challenge with a mixture of virusstrains versus single strains. At high virus doses, HaSNPV-LM2 killed H. armigera larvae significantly fas-ter (ca. 20 h) than HaSNPV-wt, but at low doses, there was no significant difference in survival timebetween the viruses. The survival time after challenge with mixed virus inoculum was significantly dif-ferent from and intermediate between that of the single viruses at high doses, and not different from thatof the single viruses at low doses. No differences in lethal dose were found between single and mixedinfections or between virus genotypes. The number of occlusion bodies produced per larva increased withtime to death and decreased with virus dose, but no significant differences among virus types were found.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

Baculoviruses occur naturally in insect populations, and somehave been developed as microbial insecticides because, due to theirspecificity, they are generally safe to non-target organisms and theenvironment. However, only a few baculoviruses have become acommercial success because of their slow speed of action, UV sen-sitivity and limited host range (Fuxa, 1991; Moscardi, 1999;Szewczyk et al., 2006; Erlandson, 2008). In the field, baculovirusesoccur as mixtures of conspecific genotypes (Miller, 1997; Hodgsonet al., 2001). This genotypic diversity in a baculovirus populationcan be the result of genetic drift from a single parent strain, dueto mutations, deletions and insertions during virus replication, orit can result from mixing and recombination of local and immi-grant baculovirus strains (Williams, and Otvos, 2005; Lauzonet al., 2005; Jakubowska et al., 2005).

ll rights reserved.

te University, Department ofiversity Park, PA 16802, USA.

Molecular engineering methods have been used to generatenovel baculovirus genotypes with improved traits, notably speedof kill. These modifications include the deletion of viral genesand the insertion of genes that express insect specific toxins ormetabolic enzymes (Bonning et al., 1992; Inceoglu et al., 2006).An important example of the former is the deletion of the bacu-lovirus egt gene. This gene encodes the enzyme ecdysteroid UDP-glucosyltransferase (egt), which modifies ecdysteroid hormonesby adding a carbohydrate moiety. These modifications renderthese hormones inactive thereby delaying or inhibiting the moltin virus-infected insects (O’Reilly and Miller, 1990, 1991). Infec-tion with an egt-deletion virus results in normal progression ofthe larval molt, and often reduces time to death of virus-infectedinsects and an attendant reduction in crop damage (Cory et al.,2004).

The persistence of genetically engineered baculoviruses in anecosystem will depend on competitive processes between virusstrains at several levels of organization and the ability of theviruses to be maintained in the ecosystem in lethal or sublethalform. Both processes within host insects (e.g. virus production)and processes at the ecosystem level, e.g. virus transmission

L. Georgievska et al. / Journal of Invertebrate Pathology 104 (2010) 44–50 45

due to encounters between larvae and inoculum, may be in-volved. These competitive interactions between recombinantand wild-type strains will determine whether recombinant bac-uloviruses will persist in an ecosystem and for how long. Giventhe high levels of heterogeneity in natural populations of baculo-viruses, competition between virus strains within hosts is highlylikely.

The cotton bollworm, Helicoverpa armigera (Hübner), is an eco-nomically important pest insect that attacks at least 35 crop and 25wild host plants (Greathead and Girling, 1982) and is susceptible toH. armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV).In Asia, India, and South Africa, H. armigera is a key pest on cotton(van Hamburg and Guest, 1997; Cherry et al., 2003). Whilst Bacill-lus thuringiensis cotton (Bt cotton) resistant to H. armigera is avail-able, it is essential that alternative control options are also studiedand optimized. This insect is highly resistant to pesticides (Armeset al., 1992; Ugurlu and Gurkan, 2008), resistance to Bt cottonmay occur (Tabashnik et al., 2003), and transgenic resistancemay not be available in minor crops that are also hosts for H.armigera.

Herein, we examined several parameters of pathogen fitness inmixed infections of wild-type and recombinant HaSNPV in H. armi-gera. Cohorts of third instars were challenged with a 1:1 mixture ofwild-type HaSNPV (HaSNPV-wt) and an egt-deletion mutant (HaS-NPV-LM2) with enhanced speed of kill, or an equivalent dose ofone of the two individual biotypes, using a range of 10 differentdoses. Data were analyzed to determine three important factorsthat impact virus fitness: lethal dose (LD50), survival time (ST50)and occlusion body (OB) yield (Hodgson et al., 2002, 2004). Whenan insect is challenged with a mixture of a fast and a slow killinggenotype of a baculovirus, different outcomes are theoreticallypossible: the time to kill could be similar to that of the fast actingvirus strain in the mixture, it could be similar to that of the slowacting virus, or it could be intermediate. We tested three alternatehypotheses for the impact of mixed infections on speed of kill: (1)time to death in larvae challenged with a virus genotype mixture issimilar to that of larvae infected with the pure egt deletion strain,(2) time to death is similar to that of larvae infected with the wild-type strain, and (3) time to death is intermediate between that oflarvae with single genotype infections.

Fig. 1. Schematic representation of HaSNPV-wt (A) and HaSNPV-LM2 (B) at the egt geneare B, BamHI, RI, EcoRI, RV, EcoRV, H, HindIII, P, PstI, S, SstI. Thick arrows indicate the oriand reverse primers on the egt and AaIT genes, respectively.

2. Materials and methods

2.1. Viruses

The baculoviruses used in this study were: (1) purified wild-type HaSNPV, isolated from H. armigera larvae from China (namedHaSNPV-G4) (Sun et al., 1998), further referred to in the text asHaSNPV-wt; (2) a recombinant HaSNPV lacking egt, further re-ferred to as HaSNPV-LM2, and (3) a 1:1 mixture of these two bac-uloviruses, further referred to as HaSNPV-mix.

HaSNPV-LM2 was generated by co-infection with HaSNPV-CXW2 DNA (-egt; +GFP) (Chen et al., 2000) and plasmid pHaLM2.Most of the egt open reading frame (ORF) in this transfer vectorwas deleted by insertion of the AaIT gene (Inceoglu et al., 2006)and a SV40 transcription termination sequence flanked by HindIIIsites (Chen et al., 2000; Fig. 1). The AaIT gene is not expressed inthis construct because the egt promoter is absent, which was con-firmed by nucleotide sequencing and by Western blot using anAaIT-specific antibody. The co-transfection was carried out usingHelicoverpa zea Hz-AM1 cells, grown in CCM3 medium supple-mented with 10% fetal bovine serum. Recombinant HaSNPV-LM2was re-isolated after three cycles of plaque purification in Hz-AM1 cells (McIntosh and Ignoffo, 1983). All viruses were amplifiedin fourth instar H. armigera reared in the laboratory on artificialdiet (Green et al., 1976). Occlusion bodies (OBs) were purified frominfected larvae by homogenization and sucrose gradient centrifu-gation (Allaway and Payne, 1984). Concentration of OBs of the viralstock solutions was determined in three independent counts usingan Improved Neubauer chamber (Hawsksley, Lancing, UK) byphase-contrast microscopy (400�). Virus stocks were stored at4 �C until use.

2.2. Insects

H. armigera used in the experiments were obtained from an in-sect colony maintained at the Department of Entomology, PublicUniversity of Navarra, Pamplona, Spain. The colony was rearedcontinuously on artificial diet (Green et al., 1976) at 25 �C, 70% rel-ative humidity (RH), and a 16L:8D h photoperiod.

locus of the HaSNPV genome (Chen et al., 2000). The restriction sites indicated hereentation of the genes ph (polyhedrin), egt and p10. Thin arrows indicate the forward

46 L. Georgievska et al. / Journal of Invertebrate Pathology 104 (2010) 44–50

2.3. Bioassays: determination of lethal dose (LD50) and survival time(ST50)

Third instar H. armigera were challenged with a range of dosesof HaSNPV-wt, HaSNPV-LM2 or a 1:1 mixture of these two virusesto determine mortality and survival time. Second instar larvaeexhibiting head capsule slippage were held without food for 16 hat 25 �C. Larvae that had molted to the third instar were orallyinoculated by the droplet feeding method (Hughes and Wood,1981), by exposing the larvae to an aqueous suspension containing10% (w/v) sucrose, 0.001% (w/v) Fluorella blue (food dye), and rel-evant concentrations of OBs. The following serial dilutions of eachvirus were used: 3 � 107, 1 � 107, 3 � 106, 1 � 106, 3 � 105,1 � 105, 3 � 104, 1 � 104, 3 � 103 and 1 � 103 OBs/ml. Sun et al.(2004) determined that third instar H. armigera under the sameconditions ingested a volume of approximately 1 ll; thus, themean ingested dosages were estimated at 1, 3, 10, 30, 100, 300,1000, 3000, 10000 and 30000 OBs/larva, respectively. Only larvaethat imbibed the virus solution, as evidenced by the blue colorationof their midgut, within 10 min after exposure to the virus solutionwere transferred onto fresh artificial diet. Controls (N = 25 larvae)consisted of larvae handled in the same manner but fed virus-freesolution instead.

Inoculated larvae were reared individually at 25 �C, 79% RH, anda 16L:8D light–dark regimen; insects were monitored at approxi-mately 8 h intervals until they died or pupated. Bioassays were re-peated six times with minor variations in the range of doses used,i.e. each experiment included most of the above-mentioned dosesbut not necessarily all. The number of larvae varied with dose from25 at high doses to 70 at low doses to obtain a sufficient number ofinfected insects at each dose.

Mean lethal dose values (LD50) were determined by probit anal-ysis after a log10 transformation of viral dose, using POLO-PLUS(Russell et al., 1977). Survival times (ST50) were estimated usingthe Kaplan–Meier Product Limit estimator in JMP SAS (2008). Inaddition, Cox’s Proportional Hazards model was used to determinethe influences of virus treatment, experiment (replicate), and theirinteraction on survival at each viral dose (Collett, 1994).

Fig. 3. Survival time (ST50) of third instar H. armigera exposed to one of threeHaSNPV preparations, plotted against dose (OBs/larva). Data were analyzed as theaverage of the median survival times (ST50) across experiments separately for eachviral dose. Vertical bars represent standard errors of the means. One asteriskrepresents a significant difference between HaSNPV-wt and HaSNPV-LM2, whiletwo asterisks over a bar represent a significant difference between HaSNPV-wt andthe 1:1 mixture of HaSNPV-wt and HaSNPV-LM2. a = 0.05.

2.4. Bioassays: determination of OB yield

OB yield was determined for two of the six bioassays fromapproximately five cadavers, randomly selected for each combina-tion of dose and virus treatment. Individual cadavers were homog-enized in 0.5 ml of sterile water, filtered through a plastic filterwith a fine metal net (mesh size 120–200 lm, pore diameter

Fig. 2. Log dose–mortality responses of third instar H. armigera exposed to one of three Hor a 1:1 mixture of HaSNPV-wt and HaSNP-LM2.

70 nm) and then centrifuged at 6000g for 5 min. The supernatantwas discarded and the OB pellet was resuspended in 500 ll of ster-ile water. Virus yield was determined by counting the number ofOBs using an Improved Neubauer hemocytometer by phase-con-trast microscopy, in three independent counts. Concentrationsare reported as OBs/ml.

OB yield was analyzed using regression analysis in SPSS (SPSSInc., 2003) and Genstat (procedure REML). The REML procedurein Genstat is equivalent to an ordinary least squares regression;for Restricted Maximum Likelihood (REML), the model is fittedusing maximum likelihood instead of least squares. Yield waslog-transformed before analysis to obtain identically distributednormal errors (IDNE). Residuals were checked visually for depar-tures from the assumption of IDNE. Experiment (random) and virustreatment (fixed) were used as factors in the analysis, time to deathas covariate, while dose was alternately included as a factor (10levels) or as a covariate (1 df).

3. Results

3.1. Mortality

Mortality increased with dose for all virus treatments (Fig. 2).Mortality responses were shallow indicating considerable varia-

aSNPV preparations (OBs/larva): HaSNPV-wt, the egt-deletion mutant HaSNP-LM2,

Table 1Survival times (ST50s) of third instar Helicoverpa armigera infected with wild-type and recombinant viruses.

Dose (OBs/larva) Virus ST50s (h.p.i.)1 95% confidence interval SE df v2 P

CI low CI high

1 HaSNPV-wt 136a 136 160 6.3 2 1.70 0.4269HaSNPV-mix 144a 128 160 5.1HaSNPV-LM2 160a 136 160 7.0

3 HaSNPV-wt 128a 112 136 4.4 2 1.91 0.3839HaSNPV-mix 128a 120 136 3.2HaSNPV-LM2 136a 120 144 4.1

10 HaSNPV-wt 144a 144 152 2.3 2 1.87 0.3924HaSNPV-mix 136a 136 144 2.6HaSNPV-LM2 136a 128 136 3.3

30 HaSNPV-wt 136a 136 144 2.4 2 1.68 0.4315HaSNPV-mix 152a 136 160 2.6HaSNPV-LM2 136a 136 144 2.3

100 HaSNPV-wt 136a 136 136 1.5 2 3.02 0.2201HaSNPV-mix 136a 136 136 1.9HaSNPV-LM2 136a 136 144 1.6

300 HaSNPV-wt 136a 128 136 1.8 2 44.2 0.0001HaSNPV-mix 128a 120 136 2.1HaSNPV-LM2 120b 112 120 1.5

1000 HaSNPV-wt 120a 120 128 1.7 2 24.6 0.0001HaSNPV-mix 120a 120 128 2.1HaSNPV-LM2 112b 104 112 1.6

3000 HaSNPV-wt 120a 120 128 1.9 2 29.3 0.0001HaSNPV-mix 104b 104 112 2.0HaSNPV-LM2 96c 96 104 1.8

10,000 HaSNPV-wt 112a 104 120 1.8 2 25.9 0.0001HaSNPV-mix 96b 96 104 2.4HaSNPV-LM2 96c 96 96 1.4

30,000 HaSNPV-wt 104a 96 112 1.8 2 11.7 0.0029HaSNPV-mix 96a 96 104 2.1HaSNPV-LM2 88b 88 96 1.7

10,0000 HaSNPV-wt 96a 96 96 3.5 2 12.3 0.0022HaSNPV-mix 88b 72 88 4.3HaSNPV-LM2 80b 72 88 3.7

30,0000 HaSNPV-wt 88a 88 96 3.5 2 26.5 0.0001HaSNPV-mix 80b 72 88 5.0HaSNPV-LM2 72c 63 72 2.6

Median survival times were determined using the Kaplan Meier Product Limit estimator (Collett, 1994); h.p.i. = hours post inoculation. Different letters after ST50 values foreach dose are significantly different with a = 0.05. Chi-square results are from test of the null hypothesis that times to death were not significantly different among virustreatments at each dose.

Fig. 4. Mean differences in survival time (ST50) of third instar H. armigera challenged with HaSNPV-LM2 or a 1:1 mix of HaSNPV-LM2 and HaSNPV-wt in comparison with theST50 of larvae challenged with HaSNPV-wt alone. Viral dose is represented on the x-axis. Symbols represent the average survival time difference for six experiments for eachvirus/dose combination. Standard error bars are shown with a = 0.05.

L. Georgievska et al. / Journal of Invertebrate Pathology 104 (2010) 44–50 47

Table 2Non-parametric survival analysis of larvae inoculated with 1 of 10 viral doses. Thesurvival function was modeled using the factors of virus treatment (wild-typeHaSNPV-wt; recombinant HaSNPV-LM2 and 1:1 mix HaSNPV-wt and HaSNPV-LM2),experiment as (replicates), and their interaction (Exp. � trt).

Dose (OBs/larva) Term Log likelihood v2 df P

1 Full model 281 16.5 8 0.0361Experiments 7.7 2 0.0208Treatments 2.1 2 0.3482Exp. � trt 4.8 4 0.3090

3 Full model 859 75.5 8 0.0001

48 L. Georgievska et al. / Journal of Invertebrate Pathology 104 (2010) 44–50

tion in susceptibility among individual larvae (Ridout et al., 1993;Ben-Ami et al., 2008; Zwart et al., 2009). Within each experiment,the LD50s among virus treatments did not differ significantly fromeach other, but there were significant differences among someexperiments, so the data were not pooled for analysis. However,within each experiment, the 95% confidence intervals for the threevirus treatments overlapped. For example, in Experiment 6, theLD50s were 13 (7–24) OBs/larva, 10 (5–18) OBs/larva, and 18 (7–49) OBs/larva for HaSNPV-wt, HaSNPV-LM2, and the HaSNPV-mix, respectively.

Experiments 69.7 2 0.0001Treatments 6.2 2 0.0445Exp. � trt 9.9 4 0.0407

10 Full model 1129 107 11 0.0001Experiments 81.9 3 0.0001Treatments 0.01 2 0.9915Exp. � trt 16.9 6 0.0096

30 Full model 1482 68.1 11 0.0001Experiments 41.7 3 0.0001Treatments 1.65 2 0.4390Exp. � trt 15.8 6 0.0146

100 Full model 3591 73.2 17 0.0001Experiments 31.8 5 0.0001Treatments 1.0 2 0.5899Exp. � trt 38.5 10 0.0001

300 Full model 2832 89.2 17 0.0001Experiments 43.2 5 0.0001Treatments 34.3 2 0.0001Exp. � trt 18.8 10 0.0429

1000 Full model 2556 63.6 17 0.0001Experiments 21.4 5 0.0007Treatments 12.9 2 0.0016Exp. � trt 19.4 10 0.0359

3000 Full model 2353 92.1 17 0.0001Experiments 45.9 5 0.0001Treatments 14.9 2 0.0001Exp. � trt 188 10 0.0006

10,000 Full model 2178 87.3 17 0.0042Experiments 38.4 5 0.0001Treatments 19.3 2 0.0001Exp. � trt 32.0 10 0.0001

30,000 Full model 2002 55.5 17 0.0004Experiments 23.1 5 0.0001Treatments 6.90 2 0.0003Exp. � trt 21.6 10 0.0313

3.2. Survival time (ST50)

ST50 decreased with increasing dose for all virus treatments,from approximately 150 h at the lowest doses to approximately80 h at the highest (Fig. 3). At virus doses of 300 OBs/larva andhigher, significant differences among virus treatments were ob-served (Table 1).

At low doses (1, 3, 10, 30 and 100 OB/larva), there was no signif-icant difference in ST50 between wild-type and HaSNPV-LM2(Fig. 3, Table 1). In contrast, HaSNPV-LM2 killed insects signifi-cantly faster (8–24 h) than HaSNPV-wt at all doses greater thanand including 300 OBs/larva (Fig. 4, Table 1).

The mixture of viruses killed larvae significantly faster (8–16 h)than HaSNPV-wt at doses of 3000, 10,000, 100,000 and300,000 OBs/larva (Fig. 4, Table 1). Also, HaSNPV-LM2 killed fasterthan the virus mixture at doses of 300 OBs/larva or higher, exceptat a dose of 100,000 OBs/larva (Fig. 4, Table 1).

There was considerable variability in treatment effects on timeto death at low viral doses, which was reflected in the large stan-dard errors observed in Fig. 4 and wide confidence intervals forthe ST50s in Table 1.

When the full survival curves were compared by dose, usingvirus treatment and experimental replicate as the main effects innon-parametric survival analysis, times to death differed signifi-cantly as a function of both main effects (Table 2). Significant dif-ferences among the three virus treatments were found at dosesP300 OBs/larva. Variability among experiments in the relative ef-fect of virus treatments was reflected as significant interactionswhen survival analysis was conducted separately at each dose(Table 2).

100,000 Full model 805 10.4 5 0.0175Experiments 0.90 1 0.0654Treatments 9.1 2 0.3363Exp. � trt 0.7 2 0.0104

300,000 Full model 632 23.8 5 0.7086Experiments 5.9 1 0.0002Treatments 18.2 2 0.0144Exp. � trt 0.09 2 0.0001

3.3. Virus yield

A plot of OB yields versus virus dose suggested that the re-sponse was not linear, and the highest OB yield was attained atintermediate doses (Fig. 5). Therefore, dose was included in theanalysis as a factor with 10 levels (9 df). Virus yield varied signifi-cantly with dose across the two experiments (Wald statistic with 9df = 57; P < 0.001; Table 3A). There was also a significant differencein yield between the two replicates (F = 51; df = 1, 241; P < 0.001;Table 3 A). When survival time was included as a covariate (1 df)in the REML analysis, it explained a large proportion of the varia-tion (Wald statistic with 1 df = 70; P� 0.001; Table 3B), whilediminishing the explanatory value of dose compared to analysis3A, indicating that part of the association between dose and virusyield in Table 3A is mediated by survival time. In both REML anal-yses, the effect of virus treatment was not significant, and therewas no significant interaction between virus treatment and doseon virus yield. The positive association between survival timeand yield is illustrated in Fig. 5. A test for equality of regressionslopes for different virus treatments in different experiments gaveno significant departure from the null hypotheses that all sub-groups were characterized by the same intercept and slope param-eters (F = 1.07; df = 5, 86; P = 0.38), supporting the notion that the

relationship between survival time and OB yield is robust acrossvirus genotypes and conditions in these experiments.

4. Discussion

The survival time of larvae challenged with a mixture of a fastand slow killing genotype of HaSNPV was intermediate betweenthe survival times of larvae challenged with the two genotypesseparately. Also, survival time decreased with increasing virus dosefor all treatments. Significant differences in ST50 between virustreatments were found at doses of 300 OBs/larva or higher. Sur-vival time decreased substantially with dose, which is commonlyobserved in baculovirus infections (e.g. Milks et al., 2001), whilevariability in the estimated survival time increased at lower doses.

Fig. 5. Relationship between time to death and yield of progeny virus (OBs/larva) ofthird instar H. armigera larvae challenged with one of 10 different doses of HaSNPV-wt; recombinant HaSNPV-LM2 or a 1:1 mixture of HaSNPV-wt and HaSNPV-LM2.Standard errors are shown for each dose with a = 0.05. (A) Experiment 1, and (B)Experiment 2.

L. Georgievska et al. / Journal of Invertebrate Pathology 104 (2010) 44–50 49

This increase in variability may have multiple causes. One is thelower numbers of insects responding, even though the number ofchallenged insects was increased at low viral doses to compensatefor much lower mortality. It is also conceivable that at lower chal-lenge doses stochastic events in the infection process at the organ-ismal level cause intrinsically greater variability in response.Finally, differences in dose due to the method of droplet feedingmay have exacerbated variability, especially at the low doseswhere the value of each OB in the infection processes is maximized(i.e., the flip side of the law of diminishing returns).

Table 3REML analysis of log-transformed OB yield/larva of 3rd instar H. armigera challenged with

df

3A: Log(virus yield) as a function of experiment, virus treatment and virus doseRandom effects Mean sq

Exp. 1 8.40Units 214 0.16

Fixed effects Wald sta

Virus treatment 2 0.79Dose 9 57.6Treatment � dose 18 23.0

3B: Log(virus yield) as a function of experiment, virus treatment and virus dose, with surRandom effects Mean sq

Exp. 1 1.93Units 213 0.15

Fixed effects Wald sta

Survival time 1 70.3Virus treatment 2 3.11Dose 9 19.0

These findings support the second hypotheses we proposed; theeffect of egt on survival time depends on the quantity of gene prod-uct and is not a qualitative response to either presence or absencein the inoculum of a virus coding for egt. The quicker death follow-ing challenge with an egt-negative HaSNPV genotype confirmsfindings of Sun et al. (2004) in which another egt-deletion HaSNPVvariant, HaSNPV-CXW1, had a faster speed of kill. A similar findingwas observed by challenge of Trichoplusia ni with an egt-negativeconstruct of AcMNPV (Cory et al., 2004). The relationship betweenviral dose and survival time may be associated with the number offounders of the viral infection. At low doses, with an attendant lowmortality, the number of virions that initiate infection may be verylow (Zwart et al., 2009). Virus spread may be slowed down by anti-viral responses that may occur in the host larva, particularly at thelevel of the midgut by sloughing of infected midgut cells (Wash-burn, et al., 1998; Li et al., 2007) making it less likely that multiplefoci of infection become established. With a low number of foci ofinfection in the insect it might take longer for the virus to colonizethe host and kill it.

In previous reports, shorter ST50s resulted in lower OB yields(O’Reilly and Miller, 1991; Cory et al., 1994; Ignoffo and Garcia,1996; Burden et al., 2000; Hernandez-Crespo et al., 2001; Sunet al., 2005) and lower OB yields can result in reduced virus trans-mission (Muñoz and Caballero, 2000; Sun et al., 2005). In contrastto these findings, Hodgson et al. (2001) reported increased yield inmixed genotype infections compared with single infections and theauthors observed no difference in times to death. Here we foundthat the best predictor for the yield from virus-infected cadaverswas the time to death, with no significant influence of virus typeor mixture. To a large extent, the effect of dose on virus yield couldbe accounted for by survival time. Thus, our conclusion in this sys-tem is that the effect of mixed virus infection on virus yield wasentirely explained by the effect of survival time, i.e., the yield frommixed infections is expected to be intermediate between that ofthe pure virus variants used in the study.

In summary, detailed analysis of a set of six independent bioas-says comparing two biotypes of HaSNPV that differ in a single trait(egt) showed a high degree of variability in survival times withinand among experiments at low viral dosages, but not at intermedi-ate and high dosages. Our results imply that only at intermediateand high viral doses are there effects of mixed infections on the re-sponse of the larval host.

one of three HaSNPV preparations: HaSNPV-wt, HaSNPV-LM2 or HaSNPV-mix.

F P

uare

51 <0.001

tistic

0.673<0.0010.204

vival time as covariateuare

13 <0.001

tistic

<0.0010.2110.025

50 L. Georgievska et al. / Journal of Invertebrate Pathology 104 (2010) 44–50

Acknowledgments

We thank Li Mei (Wuhan Institute of Virology, Wuhan China)for constructing pHaLM2, Noelia Gorria from UPNA for rearingthe H. armigera used in bioassays and Dr. Oihane Simón for usefuldiscussion and comments. L.G. was supported by a PhD fellowshipfrom NWO-WOTRO (Netherlands Foundation for Advancement inTropical Research), Grant W 83-180 and by an Erasmus-Socratesgrant for a stay at UPNA, Pamplona, Spain. K.H. was supported bya senior visitor’s Grant from Wageningen University GraduateSchool PE&RC. We thank Jacques Withagen for statistical advice.

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